The present invention relates to a process for producing a protein powder and a sustained-release preparation comprising the protein powder. Further, it relates to a sustained-release preparation comprising a specific base material, and the like.
Recently, a large amount of proteins have been produced by utilizing Escherichia coli, yeasts, animal cells, or living bodies such as goat, hamsters, etc. due to developed genetic engineering and cell technology, and put to medicinal use. However, since these proteins have very high reactivity to acidic conditions and peptic enzymes, they are not absorbed by oral administration. Then, in general, they are administered subcutaneously or intramuscularly. However, they must be frequently administered because of the generally short biological half-life. The repeated injections takes a significant physical burden on patients.
For example, a growth hormone (hereafter sometimes referred to as GH), a representative hormone which is originally produced and secreted in the anterior portion of the pituitary gland, is a protein having widely diverse physiological activities such as promotion of growth in the body, metabolism of glucose and lipids, anabolism of protein, and cell proliferation and differentiation. And GH is recently produced on a large scale by utilizing Escherichia coli in genetic recombination technology field, and put to medicinal use clinically and worldwidely. However, GH must be frequently administered in order to maintain an effective blood level because of the short biological half-life. Especially, in the case of pituitary dwarfism, a daily subcutaneous administration to infants or young patients over a long period of time ranging from a few months to 10 years or more is conducted practically.
In order to deal with such specific problems of protein medicaments, various researches have been made for drug delivery systems. An example of the systems is a sustained-release preparation which lasts a release of a protein over a long period of time. JP-A 8-217691 (WO 96/07399) discloses a process for producing a sustained-release preparation which comprises a water-insoluble or slightly water-soluble polyvalent metal salt prepared by using a water-soluble peptide type of physiologically active substance and an aqueous solution of zinc chloride, etc., and a biodegradable polymer. Further, JP-A 8-503950 (WO 94/12158) discloses, as a process for producing a sustained-release preparation comprising human GH (hereafter sometimes referred to as hGH) and a biodegradable polymer, a process for producing microcapsules as porous particles by spraying an organic solvent solution of hGH and a polymer into liquid nitrogen, with biological activity retained. Furthermore, JP-A 10-504017 (WO 95/29664) discloses a process for producing sustained-release microcapsules by dispersing solid zinc carbonate, etc. in a polymer solution, and then adding a physiologically active substance (hormone, etc.) thereto to disperse the physiologically active substance and a metal cation separately in a biodegradable polymer. Although JP-A (WO 98/27980) and JP-A 10-7538 (WO 97/01331) disclose a process for producing sustained-release preparation comprising a physiologically active polypeptide, no condition for lyophilizing the physiologically active polypeptide is disclosed.
Thus, many attempts have been made for constructing drug delivery systems with retaining physiological activity of a protein. However, as problems specific for a protein having a higher-order structure, there are possible problems relating to stability of the protein which are resulted from denaturation during production steps of a preparation, denaturation due to change the protein with time in a preparation, and/or in vivo denaturation after administration, etc. Specifically, there is a possibility that problems of a sustained-release preparation such as low efficiency of protein uptake into a preparation, excess release of a drug at an initial stage of administration, difficulty in drug release control over a long period of time, low blood level of a drug after administration of a preparation, etc., remain unsolved.
However, where a protein can be prepared in the form of a finely divided powder, further improvement of stability of the protein is expected because of decrease in molecular mobility.
JP-A 4-500527 (WO 90/13285) discloses a process for producing a finely divided protein powder by spraying an aqueous solution of the protein into liquefied gas to freeze the solution and then drying.
In addition, Journal of Pharmaceutical Science, Vol. 87, p 152 (1998) reports a process for producing a finely divided protein powder by spray drying. However, this report discloses that denaturation degree is increased reversely correlating with the particle size of aqueous protein solution particles formed by spraying, and a large amount of a surfactant should be added to control it.
Further, WO 99/48519, which has been published after the priority date of the present application, discloses a process for producing a physiologically active polypeptide powder by adding a water-miscible organic solvent and/or a volatile salt to an aqueous solution of the physiologically active polypeptide and lyophilizing the solution.
Furthermore, JP-A 9-248177 discloses a process for producing dried microbial cells by dropping droplets of a microbial cell culture on a metal plate cooled below the freezing point to freeze cells quickly.
In general, a cooling rate of lyophilization is slower than xe2x88x9210xc2x0 C./min. For example, Iyakuhin no Toketsukanso (Lyophilization of Medicines) (Yoji OHOHASHI, Preparations and Machines, page 8, Jan. 15, 1988, published by Crest, Co., Ltd.) describes as follows. xe2x80x9cIn lyophilization with a normal vial, to subject to quick freezing, or to use a solution containing a saccharide at such a high concentration that it forms a glassy state is not so often encountered unless a special apparatus is used. Namely, a cooling rate is 0.3 to 5xc2x0 C./min.xe2x80x9d On the other hand, in case of spraying an aqueous solution into liquefied gas such as liquid nitrogen, etc., a cooling rate is extremely fast, e.g., faster than xe2x88x92300xc2x0 C./min. in case of liquid nitrogen.
The minus sign in a cooling rate used herein is simply intended to express cooling. Therefore, for example, the cooling rate of xe2x88x92300xc2x0 C./min. indicates that a material to be determined is cooled by 300xc2x0 C. per 1 minute and, when a material to be determined is cooled by 150xc2x0 C. in 30 seconds, the cooling rate is indicated as xe2x88x92300xc2x0 C./min. More specifically, when a material to be determined is cooled from 20xc2x0 C. to xe2x88x92130xc2x0 C. in 30 seconds, the cooling rate is indicated as xe2x88x92300xc2x0 C./min.
Like spray drying, in a process for producing a protein powder comprising spraying a solution of the protein into liquefied gas, freezing the solution and then drying, there is a high possibility that denaturation of the protein is caused. Further, since liquefied gas is used as a liquid refrigerant, large scale and expensive facilities are required for coping with heat insulation and expansion and contraction of materials of an apparatus due to difference in temperature, maintaining aseptic conditions, evacuating the liquefied gas, etc.
In addition, although a finely divided protein powder product having an average particle size of several microns can be obtained by using a large amount of a surfactant, use of the product is restricted because of the use of a large amount of a surfactant.
Then, it is desired to simply and conveniently provide a stable protein powder which retains a higher-order structure thereof without contact with liquefied gas.
The present inventors studied to solve the above problems, and found out the fact that a protein powder retaining its higher-order structure can be obtained by controlling a cooling rate for freezing a protein-containing solution, when producing the protein powder. Further, the present inventors found out the fact that a finely divided powder can be obtained by atomizing the above-obtained protein powder. In addition, the present inventors found out the fact that, when the above-obtained protein is used for the production of a sustained-release preparation, a protein entrapment ratio of the preparation, excess release of a drug at an initial stage of administration, and sustained release property can be improved.
Also, the present inventors found out the fact that a protein powder highly retaining its higher-order structure can be obtained with controlling a cooling rate by applying or dropping a protein-containing solution when freezing the solution.
Further, the present inventors found out the fact that the desired product can be obtained cheaper and more simply and conveniently by carrying out the above freezing using a shelf of a freeze-dryer normally used in lyophilization of medicaments.
Based on these findings, the present inventors accomplished the present invention.
That is, the present invention relates to:
(1) A process for producing a protein powder which comprises contacting a protein-containing solution with a refrigerant carrier, freezing the solution at a cooling rate of about xe2x88x92300 to xe2x88x9210xc2x0 C./min. and then drying;
(2) The process according to the above (1), wherein the protein-containing solution is applied to or dropped on the refrigerant carrier;
(3) The process according to the above (2), wherein a dropping fluid of about 0.1 to 40 mm diameter is applied or dropped;
(4) The process according to the above (1), wherein freezing is carried out by preventing the protein-containing solution from direct contact with a liquid refrigerant;
(5) The process according to the above (1), wherein a volatile salt or water-miscible organic solvent is added to the protein-containing solution;
(6) The process according to the above (5), wherein the volatile salt is ammonium acetate;
(7) A protein powder obtainable by the process according to the above (1);
(8) The protein powder according to the above (7), wherein the protein has a molecular weight of about 5,000 to 1,000,000 dalton;
(9) The protein powder according to the above (7), wherein the protein is selected from hormones, cytokines, hematopoietic factors, growth factors and enzymes;
(10) The protein powder according to the above (7), wherein the protein is a growth hormone or insulin;
(11) The protein powder according to the above (7), wherein the protein retains 45% or more of xcex1-helix based on the total xcex1-helix content in the protein-containing solution;
(12) A process for producing a finely divided protein powder which comprising atomizing the protein powder according to the above (7);
(13) The process according to the above (12), wherein the atomization is carried out so that a finely divided protein powder having an average particle size of about 0.5 to 20 xcexcm is obtained;
(14) A sustained-release preparation which comprises the finely divided protein powder obtained by the process according to the above (12);
(15) The sustained-release preparation according to the above (14), wherein a base material of the sustained-release preparation is a material derived from a living body or a synthetic polymer;
(16) The sustained-release preparation according to the above (15), wherein the material derived from a living body or a synthetic polymer is a biodegradable polymer;
(17) A sustained-release preparation which comprises lactic acid/glycolic acid copolymer having the molar ratio of the lactic acid/glycolic acid of 60/40 to 70/30 and a growth hormone;
(18) A process for producing a sustained-release preparation which comprises using the finely divided protein powder obtained by the process according to the above (12);
(19) Use of the finely divided protein powder according to the above (7) for manufacturing a sustained-release preparation;
(20) The process according to the above (1), wherein the protein-containing solution is not frozen by spraying;
(21) The process according to the above (1), wherein the protein-containing solution is applied or dropped at a rate of about 10 to 250 mL/5 min. per 1300 cm2 of the refrigerant carrier cooled to about xe2x88x9225xc2x0 C. or lower before application or drop;
(22) The process according to the above (1), wherein drying is carried out under reduced pressure; and
(23) The process according to the above (1), wherein freezing is carried out by using a shelf of a freeze-dryer.